Oligonucleotides are powerful modulators of cell properties at the DNA or RNA level. Oligonucleotides may be designed to base-pair to a specific segment of DNA (triplex-forming oligonucleotides) or RNA (antisense oligonucleotides), and, after binding to the desired target nucleic acid, affect transcription and/or translation with predictable downstream biochemical and functional consequences, i.e., they inhibit gene expression. The exact mechanism by which oligonucleotides act to inhibit gene expression is still not known in all cases. Recent advances in organic synthesis and purification techniques have facilitated practicability for design and synthesis of oligonucleotides.
Because oligonucleotides are designed to bind specifically to matching sequences, they possess a high degree of specificity, and have vast potential therapeutic applications. For example, the two most common problems associated with the use of anticancer and antiviral agents are drug resistance and unacceptable levels of toxicity. These are potentially overcome by the use of oligonucleotides having high degree of specificity because: (1) genes that do not contain the target sequence are theoretically unaffected by the oligonucleotides, thereby reducing the probability of toxicity; and (2) only a point mutation within the oligonucleotide binding site, not outside, can lead to drug resistance. Therefore, oligonucleotides are thought to represent a class of powerful anticancer and antiviral agents.
Inefficient intracellular bioavailability of oligonucleotides, i.e., the amount of intact oligonucleotide that is available in a form that can interact with its intended target, has been a major obstacle in the development of oligonucleotides as therapeutic agents. Key factors contributing to inefficient intracellular bioavailability are poor stability, nonspecific binding, and poor cellular and tissue uptake.
Extensive work has been done to design derivatives of oligonucleotides having improved properties. Modifications in the nucleoside, sugar backbone, terminus, or stereochemistry comprise the four main categories of derivatization; see Uhlmann and Peyman, Chem. Rev., 90: 543-584 (1990). In addition, several delivery strategies that may circumvent some of these problems are being explored; see Akhtar and Juliano, Trends in Cell Biology, 2: 139-144 (1992) and references cited therein.
The mechanism of cellular uptake is not completely understood. For example, it is unclear whether oligonucleotide uptake occurs via a previously described transport mechanism in the cell membrane, or by a unique pathway; see Wu-Pong, Pharmaceutical Tech., pgs 102-112 (October 1994). In addition, it appears that the uptake mechanism may be cell-type and/or oligonucleotide-type dependent. Id. Current evidence indicates that oligonucleotides predominantly enter cells by endocytosis. Endocytosis is typically classified as receptor-mediated endocytosis (RME), adsorptive endocytosis (AE), and fluid-phase endocytosis (FPE).
Following endocytosis, the oligonucleotides must escape from endosomal compartments in order to exert their effects in the nucleus or cytoplasm. The precise mechanism and extent of oligonucleotide transfer from endosomes to cytoplasm and/or nuclear targets remains uncertain. Possible mechanisms are simple diffusion, transient membrane destabilization, or simple leakage during a fusion event in which endosomes fuse with other vesicles such as lysosomes. In order for oligonucleotides taken up by endocytosis to be useful, their efflux from the endosomal compartments must be improved.
Various synthetic methods have been proposed in the literature to improve intracellular bioavailability. One reported approach is the covalent attachment of specific cell receptor ligands to initiate cellular uptake by receptor-mediated endocytosis; see Vestweber and Schatz, Nature, 338: 170-172 (1989). Covalent modification of oligonucleotides generally involves the use of polycations and is a laborious procedure which requires a separate synthesis and purification for each oligonucleotide tested. Polycations such as poly-L-lysine have been reported to have been used in gene transfection studies; see Felgner, Advanced Drug Delivery Reviews, 5: 163-187 (1990). However, unlike gene tranfection, where relatively few copies of DNA are needed, use of oligonucleotides as a therapeutic agent would require high levels of DNA, and covalent modification using polycations such as poly-L-lysine may be toxic to the cells.
The most popular approach currently being investigated is the use of phospholipid vesicles (liposomes) to deliver oligonucleotides to cells; see Akhtar et al., Nucleic Acids Res., 19: 5551-5559 (1991). In addition to protecting oligonucleotides from enzymatic degradation, liposomes offer the potential to control and sustain their release. Id. Efflux of oligonucleotides from liposomes appears to be slow and sustainable over a period of several days, suggesting that they may be useful in delivering oligonucleotides against targets that have a slow turnover. Id. However, as was the case with other polycations, cationic lipids and liposomes (e.g. Lipofectin.RTM.) are generally toxic to the cells and inefficient in their DNA delivery in the presence of serum; see Leonetti et al., P.N.A.S. USA, 87: 2448-2451 (1990).
There have been extensive reports on the synthesis and use of copolymers, based on vinyl alcohol with varying levels of vinylamine, in the paper industry; see European Patent Publication 337310; European Patent Publication 339371; European Patent Publication 617166; U.S. Pat. Nos. 4,774,285, 4,880,497, 4,978,427 and 5,270,379, and as flocculants; see U.S. Pat. No. 3,715,336. There are no known reports to date relating to the use of vinyl alcohol/vinylamine copolymers for cellular therapy.
The oligonucleotide modifications and/or delivery systems described in the literature have led to improved efficiency of oligonucleotide cellular uptake; however, they have yet to allow the full potential of oligonucleotide technology to be realized. Therefore, there exists the need to develop improved methods for increasing the intracellular bioavailability of oligonucleotides. It is an object of the present invention to provide such methods. It is a further object of the invention to provide compositions for use in such methods.